Generation of α-1,3-Galactosyltransferase-Deficient Porcine Embryonic Fibroblasts by CRISPR/Cas9-Mediated Knock-in of a Small Mutated Sequence and a Targeted Toxin-Based Selection System.

摘要: Contents The CRISPR/Cas9 system has enabled the editing of mammalian genomes; however, its applicability and efficiency in pig genome not been studied depth. The α-gal epitope synthesized by α-1,3-galactosyltransferase gene (GGTA1) is known as a xenoantigen obtained upon pig-to-human xenotransplantation. We here employed system-mediated knock-in endogenous GGTA1 via targeted homologous recombination (HR). Linearized donors with ~800-bp homology flanking target site [exon 4 (containing ATG) GGTA1] served template for targeting HR. Using toxin strategy to select clones lacking expression, we successfully several within 3 weeks initial transfection. These results suggest that use CRISPR/Cas9-mediated HR mutated fragment at defined loci represents an efficient achieve rapid modulation genes interest swine cells promising tool creation KO piglets.